Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry (original) (raw)
Related papers
Experimental Cell Research, 1987
Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.
The transcription cycle of RNA polymerase II in living cells
The Journal of Cell Biology, 2002
NA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: ف 75% moved rapidly, but ف 25% was transiently immobile (association t 1/2 Ϸ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1- -D -ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on R promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [ 3 H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t 1/2 Ϸ 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases.
Experimental Cell Research, 1996
1, 2]. The spatial arrangement of chromatin domains Fluorescence in situ hybridization and immunoflu-can be altered during changes in cell cycle, differentiaorescence have been used to visualize specific genomic tion, or the pathophysiological state of the cell. It has DNA sequences and proteins in interphase nuclei been proposed that highly condensed repetitive setreated with transcriptional inhibitors. The adenosine quences (constitutively heterochromatic regions) form analog 5,6-dichloro-b-D-ribofuranosylbenzimidazole a structural framework for efficient processing of nu-(DRB) and a-amanitin selectively inhibit transcription clear events [3 -5]. The multicomponent functional by RNA polymerase II at low doses. Upon exposure structures for gene transcription and transcript proto DRB or a-amanitin the fibrillar components of the cessing and the highly substructured nucleolus are ornormally compact nucleolus unravel into necklaceganized within spatially separated subnuclear (extralike structures which represent highly extended linear chromosomal) domains. While several models of the arrays of ribosomal (r)RNA genes. Similarly, blocks of functional organization of the interphase nucleus have tandemly repeated satellite DNAs dissociate into exbeen presented [2, 6-12], the mechanisms that estabtended beaded strands. Localized (euchromatic) chrolish and maintain the many varied and interrelated mosome domains and even whole chromosome territonuclear domains remain to be clarified. Here we show ries disperse throughout the nuclear interior. Treatthat transcriptional inhibitors can disrupt the topoment of cells with actinomycin D (AMD) at doses that graphic organization of the cell nucleus into functional block rRNA synthesis does not cause significant decondomains.
Rna Synthesis by Exogenous Rna Polymerase on Cytological Preparations of Chromosomes
The Journal of Cell Biology, 1973
Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.
Conditions Favoring RNA Polymerase I Transcription in Permeabilized Cells
Experimental Cell Research, 1996
synthesis and processing occur. This approach also RNA synthesis can be detected in nuclei using modi-demonstrates that transcription by RNA polymerase fied RNA precursors (Br-UTP) introduced in perme-II (RNA pol II) is organized in discrete foci scattered abilized cells. Surprisingly, RNA pol I transcripts are throughout the nucleoplasm 2]. detected only after inhibition of RNA pol II or salt en-Surprisingly, RNA polymerase I (RNA pol I) activity hancement of RNA pol I activity. By modifying a preis rarely detected simultaneously with RNA pol II acviously reported protocol, we found that RNA pol I tivity, although the nucleolus is the nuclear territory transcripts can be detected selectively or simultanepossessing the highest level of transcription (RNA pol ously with RNA pol II transcripts without any drug I transcription of ribosomal genes (rDNA) accounts for treatment. Removing glycerol from the permeabiliza-40% of the nuclear transcription in growing cells [3, tion and transcription buffers and improving the per-4]). Additional treatments are required to reveal a numeabilization using Triton X-100 revealed RNA pol I cleolar signal: addition of a-amanitin [1, 2, 5] to inhibit transcription in two cell lines (mammalian and Xeno-RNA pol II activity and enhance antibody accessibility pus) and in isolated mouse oocytes. The transcripts by decreasing the nucleolar compaction [2, 5] and (or) were most probably rRNA because they were detected addition of NH 4 SO 4 to stimulate the RNA pol I tranin the nucleoli, digested by RNase, sensitive to actinoscription rate [1].
Active RNA polymerases are localized within discrete transcription factories' in human nuclei
Journal of cell science, 1996
Nascent transcripts in permeabilized HeLa cells were elongated by ~30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre-or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated iñ 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA poly-merase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription 'factories'. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.
Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription
Molecular Biology of the Cell, 2000
This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis. Figure 8. Scheme of the revised model of nucleologenesis.
Nucleic Acids Research, 1984
Mononucleosomes obtained from cultured mouse hepatoma cells were incubated with RNA polymerase II from wheat germ. No free DNA was liberated as available templates under the experimental condition employed. Size analysis of the transcripts showed that the polymerase initiated transcription from either terminus and read through the DNA template of mononucleosomes. Sucrose density gradient centrifugation of the reaction mixture resolved mononucleosome-polymerase complexes from free materials. The complexes were characterized by the enrichment of DNA fragments containing the nucleosome linker region, the presence of H1 histone, and the increased susceptibility to DNase I. Both the complexes formed in the presence and absence of precursor nucleotides were susceptible. These suggest that RNA polymerase II prefers to bind to the linker region, and the polymerase-bound nucleosomes are structurally altered. The data were discussed in context with possible mechanisms of transcription of the nucleosome structure.
Biochimica et biophysica acta, 1981
Two forms of RNA polymerase II were released from rat liver chromatin by micrococcal nuclease digestion of the nuclei. One from behaved like a free RNA polymerase II and the other like a complex with other nuclear components. Both forms of RNA polymerase II activity were recovered in the 0.16 M NaCl-soluble fraction of the nuclear digest, and the complexed from the RNA polymerase II could transcribe its endogenous template under conditions permitting only of elongation of the RNA synthesis. The RNA polymerase II complex was further purified by gel filtration chromatography and column electrophoresis. Analysis of protein and DNA of the partially purified complex suggested that the RNA polymerase II was bound to mono- or dinucleosomes carrying some characteristic nonhistone proteins. Furthermore, in experiments on tissues from starved rats, the two forms of RNA polymerase II were found to originate from different functional states of the chromatin-bound enzyme in vivo.